Gold nanoparticle (GNP) interaction with hen egg white lysozyme (Lyz) has been investigated by many groups in order to understand protein mediated aggregation of GNPs and the underlying mechanism of aggregation. In this article, we have studied the interaction of citrate-capped GNPs of 16, 28, 41, and 69 nm sizes with Lyz by the non-destructive label-free second harmonic light scattering (SHLS) technique at physiological pH in phosphate buffer. The surface sensitivity of the nonlinear optical SHLS technique is very high and we have looked at the GNP-Lyz interaction at nanomolar concentrations. We have followed the increase in the SHLS intensity of GNPs as a function of the added concentration of Lyz in small aliquots. The SH intensity profile exhibits saturation behaviour and was fitted with a modified Langmuir adsorption model which yielded the binding constant (Kb), the binding stoichiometry (nsat) at saturation and the free energy change (ΔG) in the adsorption process. The free energy change was further decomposed into changes in the enthalpy (ΔH) and entropy (ΔS) of adsorption by carrying out temperature dependent SHLS measurements in a specially designed cell. The thermodynamic quantities extracted from the measurements show that the binding is exothermic (ΔH < 0) as well as spontaneous (ΔS > 0). We find that the first step in the adsorption of Lyz on the GNP surface is nanoparticle protein corona (NP-PC) formation driven predominantly by electrostatic attraction. In the second step of adsorption, the adsorbed lysozymes on the surface form a bridge between two or more GNPs leading to the latter's aggregation, which is the main reason for the enhancement of the SH scattering signal. Although the interaction between the GNPs and Lyz is driven by strong electrostatic attraction, the thermodynamic quantities reported here indicate that the protein is physisorbed on the nanoparticle surface. We have also demonstrated that SHLS provides a new tool for full thermodynamic characterization of protein adsorption on metal nanoparticles at ultralow concentrations.

OBJECTIVE

To screen the radiosensitivity-related genes of colorectal cancer cells.

METHODS

Gene expression profiles of two different radiosensitivity cells(colorectal cancer cell line Lovo and SW480) were obtained by cDNA array and the differences of gene expression profiles between the two cells were analyzed.

RESULTS

Genes of more than 2-fold expressive differentiation were screened. In Lovo cells, 908 up-regulated genes were found, including higher expression genes CEACAM5, THBS1, SERPINE2, ARL7, HPGD, while 1312 genes were down-regulated. In SW480 cells, higher expression genes were SCD, NQ01, LYZ, KRT20 and ATP1B1.

CONCLUSIONS

Gene profiles can reflect the radiosensitivity of colorectal cancer cells, which will provide the choice for the further study of radiosensitivity in colorectal cancer.

The highly ordered mesoporous silicas with elaborately controlled microenvironment were synthesized via covalent incorporation of long-chain polymers (M(w) = 2000 g mol(-1)) bearing specific hydrophilic/hydrophobic balance. The microenvironment (hydrophilicity/hydrophobicity) of the mesoporous silicas was quantitatively determined by gas adsorption experiments and investigated by lysozyme (LYZ) adsorption. The relative activity of lipase from Pseudomonas cepacia (PCL) encapsulated in the mesoporous silica with moderate hydrophobic microenvironment (hereafter denoted as MHM) reaches up to 281% compared with the free PCL, notably higher than that of PCL accommodated in the mesoporous silicas with hydrophilic or strong hydrophobic microenvironment (20.7-26.2% relative to the free PCL). Moreover, PCL entrapped in the nanochannels with MHM affords the highest initial rate in the kinetic resolution of (R,S)-1-phenylethanol relative to other immobilized PCL. The above results suggest that the MHM could render the active center of PCL entirely exposed to the substrates without interrupting its native conformation in the "interfacial activation". In addition, the nanochannels with MHM could markedly improve the thermal stability of PCL (preserving nearly 60% of the initial activity after the incubation at 70 °C for 2 h) and facilitate the recycling of the immobilized PCL in both aqueous and organic media. Our work demonstrates that the subtle modulation of the microenvironment of mesoporous silicas for enzyme immobilization designates a very promising strategy to fabricate the highly active and stable heterogeneous biocatalysts for industrial application.

The combination of the molecular imprinting technology and porous materials is a promising way to obtain high-efficient selective adsorption and separation materials for bioactive macromolecules. In this work, we developed a novel approach to prepare near-infrared (NIR)-light-response inverse-opal lysozyme (Lyz)-imprinted polydopamine/polypyrrole (IO-PDA/PPy-MIP) composite microspheres using micron-sized SiO₂ colloidal crystal microspheres as the sacrificed template. The pore size of the IO-PDA/PPy-MIP microspheres can be tuned from 200 to 800 nm by the size of silica nanoparticles which self-assemble to form the template SiO₂ colloidal crystal microspheres. The IO-PDA/PPy-MIP microspheres show a rapid selective adsorption ability for Lyz due to the inverse-opal macroporous structure. The adsorption capacity exceeds 800 mg/g within 20 min, and the imprinting factor is as high as 24. The bound Lyz molecules can be released rapidly from IO-PDA/PPy-MIP microspheres triggered by the irradiation of NIR laser and remain enough bioactivity to decompose Escherichia coli efficiently. The prepared IO-PDA/PPy-MIP microspheres also exhibit excellent structure stability and good recyclability. The adsorption capacity can remain up to 90% of the initial value after 5 times recycle. This work provides not only a method to prepare novel NIR-light-response inverse-opal macroporous molecularly imprinted microspheres, but also a new perspective on the design of selectively separation materials for the fast, high-efficient recognition and separation of biomacromolecules.

A molecularly imprinted inorganic-organic hybrid monolithic capillary column (MIP hybrid monolith) was synthesized by one-pot process and its application in selective recognition and capture of lysozyme (Lyz) from complex biological samples was described for the first time. Due to a combination of rigid silica matrices and flexible organic hydrogels in one-pot process, stable and accessible recognition sites in the as-prepared MIP hybrid monolith could be obtained after the removal of template protein, which facilitated the rebinding of template and provided good reproducibility and lifetime of use. The morphology, permeability, and pore properties of the as-prepared MIP hybrid monolith were characterized and a uniform monolithic matrix with high surface area and large through-pores was observed. The recognition behavior of MIP and non-imprinted (NIP) hybrid monolith was evaluated by separating template protein from unfractionated protein mixture and the result indicated that the MIP hybrid monolith has much higher affinity toward the template protein than NIP hybrid monolith. High imprinted factor (IF) and separation efficiency could be obtained. In addition, the practicality of the Lyz-MIP hybrid monolith was further evaluated by selective separation of Lyz from egg white and capture of Lyz from human serum by adopting it as an in-tube solid phase microextraction (in-tube SPME), and the good results demonstrated its potential in proteome analysis.

BACKGROUND

Toll-like receptor 4 (TLR4) has been implicated in inflammation-induced bone destruction in various chronic bone diseases; however, its direct influence on bone healing is not well understood. The authors' previous study showed accelerated bone healing with higher osteoclastogenesis gene expression in toll-like receptor 4 knockout mice (TLR4). This study aimed to further elucidate the underlying cellular mechanisms during fracture healing by generating a myeloid cell-specific toll-like receptor 4 knockout model (Lyz-TLR4 mice).

METHODS

Calvarial defects, 1.8 mm in diameter, were created in wild-type, TLR4, and Lyz-TLR4 mice. Bone healing was investigated using micro-computed tomography and histologic, histomorphometric, and immunohistochemistry analyses. Primary bone marrow-derived cells were also isolated from wild-type, TLR4, and Lyz-TLR4 mice to measure their osteoclast differentiation and resorption properties.

RESULTS

A similar faster bone healing response, with active intramembranous bone formation, intense osteopontin staining, and more osteoblast infiltration, was observed in TLR4 and Lyz-TLR4 mice. Tartrate-resistant acid phosphatase staining showed more osteoclast infiltration in Lyz-TLR4 mice than in wild-type mice at day 7. Primary bone marrow-derived cells isolated from TLR4 and Lyz-TLR4 mice presented enhanced osteoclastogenesis and resorption activity compared with those from wild-type mice. Comparable M0, M1, and M2 macrophage infiltration was found among all groups at days 1, 4, and 7.

CONCLUSIONS

This study revealed that inactivation of toll-like receptor 4 in myeloid cells enhanced osteoclastogenesis and accelerated healing response during skull repair. Together with the role of toll-like receptor 4 in inflammation-mediated bone destruction, it suggests that toll-like receptor 4 might regulate inflammation-induced osteoclastogenesis under different clinical settings.

Circadian rhythm provides organisms with an internal system to maintain temporal order in a dynamic environment. This is typified by a 24-h cycle for a number of physiological processes, including immunity. The present study characterized the humoral and mucosal defense molecules and their dynamics during a light-dark (LD) cycle in juvenile permit, Trachinotus falcatus. All studied defense molecules were constitutively identified in serum and skin mucus. Serum generally exhibited higher levels of these defenses than skin mucus, with the exception of anti-protease (ANTIPRO). The difference in ANTIPRO, lysozyme (LYZ), esterase (ESA) and catalase (CAT) levels between serum and skin mucus was not affected by the phase of the daily cycle. However, a clear phase-dependent difference was observed in protease (PRO), globulin (GLOB), myeloperoxidase (MPO), alkaline phosphatase (ALP) and glutathione peroxidase (GPX) levels. Activities of ALP and GPX displayed significant daily rhythmicity in both serum and skin mucus. Circadian profile of ALP was identical in both biofluids, but an antiphasic feature was exhibited by GPX. GLOB and MPO levels also exhibited significant daily oscillation but only in serum with acrophases registered at ZT 14.5 and 6.15, respectively. Mucus PRO and serum ANTIPRO demonstrated significant temporal variations during a daily cycle albeit not rhythmic. Cluster analysis of the defense molecules in serum and skin mucus revealed two different daily profiles suggesting a possibility of distinct circadian control between humoral and mucosal immunity. These observations indicate that LD cycle had a remarkable impact in the defense molecules characterizing the humoral and mucosal immunity in permit. Daily rhythmic patterns of these defense molecules contribute to our understanding of the barely explored interplay of immunity and circadian rhythm in teleost fish. Lastly, the results could be useful in developing aquaculture practices aiming at modifying the immune functions of permit for improved health.

To investigate the protective and destructive effects of sodium dodecyl sulfate (SDS) in thermal denaturation of proteins, we carried out twelve independent atomistic molecular dynamics (MD) simulations of bovine serum albumin (BSA) and hen egg-white lysozyme (LYZ) in pure water and SDS solutions at 25 and 80 °C, using SDS concentrations of 0.01, 0.05, 0.1 and 1 M. In the case of the BSA in pure water and SDS solutions, it was found that its helicity decreased from 67.02% in reference structure to 35% in pure water at 80 °C due to thermal denaturation, whereas it increased to 49.34, 52.36 and 54% at 0.01, 0.05 and 0.1 M SDS, respectively, owing to the SDS protective effect. In 1 M SDS, however, the surfactant's protective effect was weak, and consequently the helicity of the BSA decreased to 47.01%. In contrast, no protection by SDS was observed for the LYZ in SDS solutions as the loss of its helices increased with SDS concentration from 0.01 to 1 M. In attempt to interpret the SDS effects molecularly, we calculated the diffusion coefficients of SDS in the protein solutions. The calculated values were found to decrease with increasing SDS concentration in the BSA solutions, but to increase with SDS concentration in the LYZ solutions. The decrease or increase in the diffusion coefficient of SDS was attributed to the net negative or positive charge on the proteins at neutral pH, indicating that electrostatic repulsions or attractions affect diffusivity significantly and can moderate SDS-proteins non-covalent interactions.

The double template surface-imprinted polymer (Bi-MIP)) was synthesized by atom transfer radical polymerization (ATRP) in aqueous media, using bovine serum albumin (BSA) and lysozyme (Lyz) as the template prteis, X-isopropylacrylamide (NIPAAm) and acrylamide (AAm) as the monomers and X-3-(dimethylamino) propyl-methacrylamide (DMAPMA) as the assistant of basic functional monomner. The ATHP possessed the mild reaction conditions and can be initiated at room temperature without heating and ultraviolet radiations. The preparation conditions of imprinted polymer were optimized, and the content of DMAPMA as the assistant of basic functional monomer was investigated. The results showed that Bi-MIP exhibited the good adsorption capacity and selectivity in single protein solution and in mixed protein solution when the volume of DMAPMA in the preparation process of Bi-MIP was 20 µL. The rebinding capacity of Bi-MIP was evaluated according to adsorption isotherm of the imprinted polymer. The Langmir adsorption model was employed to describe the isotherms and revealed that Bi-MIP exhibited the maximum rebinding to 13SA and Lyz at 10.2 mg g and 19.2 mg/g, respectively. And Bi-MIP had good imprinting effect and adsorption capacity to templates BSA and Lyz from egg white and bovine serum samples. This will provide a new way for specific recognition of the double multiple target proteins in the complex biological samples.

Ionic liquid (IL)-assisted sodium dodecyl sulfate polyacrylamide gel electrophoresis (ILs-SDS-PAGE) was presented to improve protein separation. ILs were employed during the preparation process of polyacrylamide gel, then the modified gel was used for commercial protein marker, binary bovine serum albumin/lysozyme (BSA/Lyz) and human serum separation. The influence of ionic liquid concentration, cation alkyl chain length, cation and anion types on proteins separation were investigated. The results showed that ILs played a role in improving some protein separation, and ILs-SDS-PAGE provided higher resolution and separation efficiency than ordinary SDS-PAGE for low and middle relative molecular mass proteins in human serum. In addition, the principle of ILs-SDS-PAGE was discussed and the comparison of ILs-SDS-PAGE with ordinary SDS-PAGE and Native PAGE was made.

The interaction of drospirenone (DP) with human serum albumin (HSA)/lysozyme (LYZ) was investigated using different optical techniques and molecular models. Results from the emission and time resolved fluorescence studies revealed that HSA/LYZ emission quenching with DP was initiated by static quenching mechanism. The LYZ-DP system was more easily influenced by temperature than the HSA-DP system. Displacement experiments demonstrated that the DP binding site was mainly located in site 1 of HSA. Based on the docking methods, DP was mainly bound in the active site hinge region where Trp-62 and Trp-63 are located. Conformation study showed that DP had different effects on the local conformation of HSA and LYZ molecules.

Protein adsorption plays a key role in bone repair and regeneration by affecting cell behavior. In this study, TiO₂ nanofibers (TiO₂ NFs) with different structures, including anatase TiO₂ nanofibers (A-NFs), anatase TiO₂ nanofibers with beads (B-NFs), anatase-rutile TiO₂ nanofibers (AR-NFs) and rutile TiO₂ nanofibers (R-NFs), were prepared by electrospinning method. Bovine serum albumin (BSA) and lysozyme (LYZ) were used to explore the adsorption behaviors of TiO₂ NFs and then the effects of materials with protein on bone marrow mesenchymal stem cells (MSCs) were studied. Pure titanium metal (PT) was used as control. The results displayed that the adsorption amounts of BSA on samples were B-NFs > AR-NFs > A-NFs ≈ R-NFs > PT, and that for LYZ were B-NFs > AR-NFs > R-NFs > A-NFs > PT. The conformation of proteins changed remarkably when they were adsorbed on meterials. Soaking the TiO₂ NFs with and without protein into SBF revealed that the BSA and LYZ on B-NFs, A-NFs and AR-NFs could accelerate the HA deposition on its surface, but it had no promoting effect on HA deposition on B-NFs. MTT and PCR tests showed that the BSA and LYZ adsorbed on materials could promote the proliferation and osteogenic differentiation of MSCs to different degrees due to their different adsorption amount and conformation changes on different TiO₂ NFs. The current work demonstrated that the surface properties and crystal structure of TiO₂ NFs could influence the adsorption behavior and conformational change of BSA and LYZ, and then further regulate MSCs biological behavior.

To search immune defense proteins in skin mucus of Japanese flounder fed with a diet containing high concentration of ascorbic acid, we carried out 2D-PAGE and compared the resolved pattern of proteins between control group that fed commercial diet and ascorbic acid supplemented group (AsA group) fed a diet supplemented with high concentration of ascorbic acid (2,000mg/kg) for 7days. The results revealed that there were many proteins exhibited distinct increase in AsA group. Among them, 6 regions that showed a dramatic elevation were chosen for protein identification using LC-MS/MS analysis and Mascot database search. Six proteins were identified, i.e. serotransferrin (Sero), transferrin (Trans), warm temperature acclimation-related 65 kDa protein (Wap65), complement component c3 (C3), hemoglobin beta-A chain (Hbß) and apolipoprotein A-1 (Apo). Quantitative RT-PCR analysis showed that the mRNA level of Hbß in epidermis of AsA group gave much higher increase (11.6 folds) than control group; the levels of Sero/Trans, Wap65, C3 and Apo showed no apparent difference between the two groups. The mRNA levels of wap65 and c3 in the liver and Apo in the kidney of AsA group exhibited significant increase in comparison to control group. In the case of secreted immunoglobulin M (IgM) and lysozyme (lyz), no difference of the mRNA levels of IgM in epidermis, gill, kidney, spleen and intestine, and lyz in epidermis, gill, spleen and intestine, was observed. The results of in situ hybridization confirmed the elevation of Hbß mRNA level in the epidermis tissue of AsA group. Our present study provided additional evidence showing the effectiveness of AsA in activating innate immune defense system in skin mucosal tissue of fish.

Keywords: Japanese flounder; ascorbic acid; immune factors; immunostimulants; proteome analysis; skin mucus.

Purpose: There is considerable controversy on the role of genetics, mechanical and environmental factors, and, recently, on subclinical infection in triggering inflammaging leading to disk degeneration. The present study investigated sequential molecular events in the host, analyzing proteome level changes that will reveal triggering factors of inflammaging and degeneration.

Methods: Ten MRI normal disks (ND) from braindead organ donors and 17 degenerated disks (DD) from surgery were subjected to in-gel-based label-free ESI-LC-MS/MS analysis. Bacterial-responsive host-defense response proteins/pathways leading to Inflammaging were identified and compared between ND and DD.

Results: Out of the 263 well-established host-defense response proteins (HDRPs), 243 proteins were identified, and 64 abundantly expressed HDRPs were analyzed further. Among the 21 HDRPs common to both ND and DD, complement factor 3 (C3) and heparan sulfate proteoglycan 2 (HSPG2) were significantly upregulated, and lysozyme (LYZ), superoxide dismutase 3 (SOD3), phospholipase-A2 (PLA2G2A), and tissue inhibitor of metalloproteinases 3 (TIMP-3) were downregulated in DD. Forty-two specific HDRPs mainly, complement proteins, apolipoproteins, and antimicrobial proteins involved in the complement cascade, neutrophil degranulation, and oxidative-stress regulation pathways representing an ongoing host response to subclinical infection and uncontrolled inflammation were identified in DD. Protein-Protein interaction analysis revealed cross talk between most of the expressed HDRPs, adding evidence to bacterial presence and stimulation of these defense pathways.

Conclusions: The predominance of HDRPs involved in complement cascades, neutrophil degranulation, and oxidative-stress regulation indicated an ongoing infection mediated inflammatory process in DD. Our study has documented increasing evidence for bacteria's role in triggering the innate immune system leading to chronic inflammation and degenerative disk disease.

Keywords: Disk degeneration; Disk proteome; Dysbiosis; Host-defense response proteins; Inflammaging.

ABSTRACT The lysozyme from Erwinia amylovora phage PhiEa1h was investigated for its ability to inhibit growth of bacteria and compared with the lysozyme from Escherichia coli phage T4. The assays to measure lysozyme activity included cell lysis and growth inhibition of bacteria. Bacterial strains with kanamycin resistance were not affected by lysates containing the PhiEa1h-enzyme. The titer of Micrococcus luteus but not of Erwinia amylovora was diminished by cell extracts containing T4 lysozyme. In contrast, PhiEa1h lysozyme preferentially inhibited E. amylovora, exceeding the T4 lysozyme activity at least one million-fold. Spherical cells were formed after application to E. amylovora similar to lyz-gene expression in Escherichia coli. Heating of cell extracts destroyed the murami-dase activity, but retained an antibacterial activity. Other plant-associated bacteria related to Erwinia amylovora also were inhibited for growth when cell extracts with PhiEa1h lysozyme were applied to soak pear slices and potato slices. Ooze formation and soft rot caused by E. amylovora or E. carotovora subsp. atroseptica, respectively, were strongly reduced and the PhiEa1h lysozyme was more efficient compared with extracts containing T4 lysozyme.

With increasing utilization of silver nanomaterials, growing concerns are raised on their deleterious effects to the environment. Once discharged in aquatic environment, the interactions between silver nanowires (AgNWs) and proteins may significantly affect the environmental behaviors, fate and toxicities of AgNWs. In the present study, three representative model proteins, including ovalbumin (OVA), bovine serum albumin (BSA) and lysozyme (LYZ), were applied to investigate the impacts of the interactions between proteins and AgNWs on the transformations (oxidative dissolution and sulfidation) of AgNWs in aquatic environment. Fluorescence spectroscopy and isothermal titration calorimetry analyses indicated that there was very weak interaction between OVA or BSA and AgNWs, but there was a strong interaction between the positively charged LYZ and negatively charged AgNWs. The presence of LYZ not only reversed the surface charge of AgNWs, but also resulted in the break-up of the nanowire structure and increased the reactive surface area. The positively charged surface of AgNWs in the presence of LYZ favored the access of sulfide ions. As a consequence, the kinetics of oxidative dissolution and sulfidation of AgNWs were not affected by OVA and BSA, but were significantly facilitated by LYZ. The results shed light on the important roles of electrostatic interactions between AgNWs and proteins, which may have important implications for evaluating the fate and effects of silver nanomaterials in complicated environment.

In this study, a series of well-controlled bottle-brush polymers, poly(methylacrylic acid)-g-poly(2-methyl-2-oxazoline) with monothiol-terminated group ((PMAA-g-PMOXA)-SH) were synthesized by using reversible addition-fragmentation chain transfer (RAFT) polymerization and cationic ring-opening polymerization (CROP). (PMAA-g-PMOXA)-SH were grafted to the surface of gold sensors in in situ aminolysis reactions. Cyclic voltammetry (CV), X-ray photoelectron spectroscopy (XPS), variable angle spectroscopic ellipsometry (VASE), water contact angle (CA), and atomic force microscopy (AFM) were used to characterize the copolymer modified gold sensor. The protein-resistant properties were investigated by surface plasmon resonance (SPR), and platelet adhesion was observed by scanning electron microscopy (SEM). Compared with the bare gold sensor, the (PMAA₂₀-g-PMOXA₁₂)-SH modified gold sensor can reduce the levels of fibrinogen (Fg), bovine serum albumin (BSA), and lysozyme (Lyz) adsorption by 96.5 ± 3.1%, 85.8 ± 5.7%, and 49.4 ± 1.6%, respectively. Meanwhile, the (PMAA₂₀-g-PMOXA₁₂)-SH modified gold sensor also possesses excellent resistance to platelet adhesion. All these data demonstrate that this simple method is feasible, and that a bottle-brush (PMAA-g-PMOXA)-SH modified gold sensor has potential applications in biosensors and biomedical areas.

A classification of [Formula: see text] contravariant Minkowski valuations on convex functions and a characterization of the projection body operator are established. The associated LYZ measure is characterized. In addition, a new [Formula: see text] covariant Minkowski valuation on convex functions is defined and characterized.

Quantum dots (QDs) have significant potentials in biomedical applications of bioimaging and biosensing. Spontaneous adsorption of proteins on QDs surface is a common phenomenon, which occurred to serum proteins in biological samples, and has been observed to enhance QDs fluorescence. In this study, fluorescence alteration of 3-mercaptopropionic acid (MPA) capped CdSe quantum dots by four individual biomarker proteins was investigated. By monitoring the fluorescence emission of QDs, the biomarker protein adsorbed spontaneously on QDs surface was recognized and quantified. When alpha fetoprotein (AFP) or heat shock protein 90 alpha (HSP90α) were present, the QDs became brighter. The presence of cytochrome C (CytoC) or lysozyme (Lyz) made the QDs dimmer first, and then brighter. Within five minutes response time all four biomarker proteins were detected individually with the estimated detection limit in the range of 1-10 ng/mL and good linear dynamic ranges. The results suggested that the fluorescence of QDs was responsive to not only serum proteins but also biomarker proteins. The fluorescence response was able to correlate quantitatively with the amount of biomarker proteins in relatively low concentrations. These results provide more information to understand QDs and support their applications in biomedical fields.

The binding of the iminium and alkanolamine forms of chelerythrine to lysozyme (Lyz) was investigated by spectroscopy and docking studies. The thermodynamics of the binding was studied by calorimetry. Spectroscopic evidence suggested that Trp-62 and Trp-63 in the β-domain of the protein are closer to the binding site; moreover, the binding site was at a distance of 2.27 and 2.00 nm from the iminium and alkanolamine forms, respectively, according to the Forster theory of non-radiation energy transfer. The equilibrium binding constants for the iminium and alkanolamine forms at 298 K were evaluated to be 1.29 × 10(5) and 7.79 × 10(5) M(-1), respectively. The binding resulted in an alteration of the secondary structure of the protein with a distinct reduction of the helical organization. The binding of iminium was endothermic, involving electrostatic and hydrophobic interactions, while that of alkanolamine form was exothermic and dominated by hydrogen bonding interactions. Docking studies provided the atomistic details pertaining to the binding of both forms of chelerythrine and supported the higher binding in favour of the alkanolamine over the iminium. Furthermore, molecular dynamics study provided accurate insights regarding the binding of both chelerythrine forms in accordance with the experimental results obtained. Chelerythrine binding pocket involves the catalytic region and aggregation prone K-peptide region, which are sandwiched between one another. Overall, these results suggest that both the forms of the alkaloid bind to the protein but the neutral form has higher affinity than the cationic form.

GdmCl (6 M) unfolded lysozyme was previously shown to refold via kinetically partitioned pathways (Kiefhaber in Proc Natl Acad Sci 92:9029-9033, 1995). About 80% of the unfolded lysozyme molecules refold on a slow pathway with well-populated intermediates. The remaining 20% of denatured lysozyme refold on a fast track without detectable intermediate. This kinetic heterogeneity has been proposed to originate from the collapsed state of lysozyme folding. Using the method of disulfide scrambling, we demonstrate in this report that these two populations of unfolded lysozyme can be isolated and analyzed separately. GdmCl (6 M) denatured lysozyme actually comprises two major populations of unfolded isomers, namely X-LYZ-a and X-LYZ-b with molar ratio of about 80:20. X-LYZ-a and X-LYZ-b exist in equilibrium in the unfolded state. Their disulfide structures and CD properties indicate that X-LYZ-a is more extensively unfolded than X-LYZ-b. Refolding experiments using the method of disulfide scrambling also show that folding kinetics of X-LYZ-a is about 8-10 times slower than that of X-LYZ-b and folding intermediates of X-LYZ-a is far more heterogeneous than that of X-LYZ-b. The results highlight the implication of the conformational heterogeneity of 6 M GdmCl denatured proteins for the interpretation of the initial stage of protein folding mechanism.

Genes coding for lysozyme-inhibiting proteins (Ivy) were cloned from the chromosomes of the plant pathogens Erwinia amylovora and Erwinia pyrifoliae. The product interfered not only with activity of hen egg white lysozyme, but also with an enzyme from E. amylovora phage ΦEa1h. We have expressed lysozyme genes from the genomes of three Erwinia species in Escherichia coli. The lysozymes expressed from genes of the E. amylovora phages ΦEa104 and ΦEa116, Erwinia chromosomes and Arabidopsis thaliana were not affected by Ivy. The enzyme from bacteriophage ΦEa1h was fused at the N- or C-terminus to other peptides. Compared to the intact lysozyme, a His-tag reduced its lytic activity about 10-fold and larger fusion proteins abolished activity completely. Specific protease cleavage restored lysozyme activity of a GST-fusion. The bacteriophage-encoded lysozymes were more active than the enzymes from bacterial chromosomes. Viral lyz genes were inserted into a broad-host range vector, and transfer to E. amylovora inhibited cell growth. Inserted in the yeast Pichia pastoris, the ΦEa1h-lysozyme was secreted and also inhibited by Ivy. Here we describe expression of unrelated cloned 'silent' lyz genes from Erwinia chromosomes and a novel interference of bacterial Ivy proteins with a viral lysozyme.

This study was carried out to investigate the effects of dietary vitamin A (VA) on growth performance, antioxidant capacity, digestion, intestinal immune response, and mRNA expression of intestinal tight junction proteins for juvenile hybrid grouper (Epinephelus fuscoguttatus ♀ × Epinephelus lanceolatus ♂). Six isonitrogenous and isolipidic experimental diets were formulated to obtain VA levels (317, 1136, 2038, 4142, 7715, 15204 IU/kg diet, respectively). The triplicate groups of fish (average weight of 9.01 ± 0.27 g) were fed twice daily (8:00 and 16:00) for 7 weeks. Based on the broken-line analysis model of WG and LYZ activity, the dietary VA requirement of hybrid grouper were estimated to be 2688.58 and 4096.36 IU/kg diet. The results showed that VA deficiency or excess could reduce Weight gain, specific growth rate, and protein efficiency ratio, and increase feed conversion ratio and hepatosomatic index (P < 0.05). In addition, VA deficiency could reduce the serum activities of acid phosphatase (ACP), superoxide dismutase, and total antioxidant capacity and increase the malondialdehyde content (P < 0.05). VA deficiency also could reduce intestinal activities of ACP, alkaline phosphatase, lysozyme, complement 3, complement 4 contents, and activities of alpha-amylase, lipase, and trypsin (P < 0.05). Meanwhile, VA deficiency could reduce villus height in proximal intestine (PI) and mid intestine (MI), as well as muscle thickness in PI and distal intestine (DI) (P < 0.05). Moreover, VA deficiency could down-regulated antimicrobial peptides (β-defensin, Hepcidin [not in MI and DI], Epinecidin), anti-inflammatory cytokines (interleukin 10 and transforming growth factor β1 [not in DI]), tight junction proteins (occluding and claudin3) mRNA levels in the PI, MI and DI, and up-regulated pro-inflammatory cytokines (tumor necrosis factor α [not in MI] and interleukin 1β [not in MI]), signaling molecules c-Rel and p65 (P < 0.05). Collectively, VA deficiency could reduce growth performance because of a negative effect on intestinal health by depressing digestive abilities, intestinal morphology, immunity and tight junction function in the intestine.

Keywords: Antioxidant capacity; Digestion; Growth performance; Intestinal immune response; Tight junction proteins; Vitamin A.

Conformational changes of proteins have an influence on their biological activity, so as to affect their use efficiency. However, the conformation of proteins is typically measured in a mixture containing the adsorbed protein, free protein and adsorbing material, which does not truly reflect the influence of the material on protein conformation. In this study, Fe3O4/carboxymethylated chitosan (Fe3O4/CMCS) nanoclusters with unique superparamagnetism were utilized as the separation carrier to study the conformational changes of the adsorbed and the free proteins. Four representative proteins with different molecular weights and isoelectric points, lysozyme (LYZ, 13.4 kDa; pI 10.8), bovine hemoglobin (BHB, 64.5 kDa; pI 6.9), apo-transferrin (TRT, 80 kDa; pI 5.9) and bovine serum albumin (BSA, 68 kDa; pI 4.8), were selected as model proteins to investigate the influences of material coating with/without metal ions and environmental factors including pH and ion strength, on the conformational behaviors of the adsorbed or free proteins. This study was aimed at providing a platform for an improved reflection of the conformational changes of proteins and has a potential to guide immobilization and separation of proteins.